(629f) Engineering a 3D Scaffold Based Hypoxic System As a Tool for Radiation Response Studies for Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a cancer of unmet clinical needs. Non-specific symptoms and late diagnosis, high metastatic occurrence and treatment resistance elucidates to extraordinarily low survival rates.^[1-4] More specifically, PDAC has a 5-year relative survival rate of just 9%.^[4] This figure has failed to improve inline with other cancers for over 50 years.^[6]Moreover, predictions suggest that this disease will rise to one of the most lethal cancers in coming decades.^[7] PDAC treatment resistance is highly influenced by the tumour microenvironment (TME). The PDAC TME is a unique heterogeneous composition of biological, biophysical, biomechanical and biochemical milieus that directly influence tumour survival and impact patient response to treatment. More specifically, pancreatic cancer cells activate pancreatic stellate cells that secrete high amounts of extracellular matrix (ECM) proteins, this accumulation of ECM increases tumour stiffness, creating extensive fibrosis/ dense desmoplasia, this results in blood vessel collapse, causing impaired intravenous drug delivery and expanses of low oxygen, known as hypoxia, highly influencing treatment response to radiotherapy.^[8,9] This unique ecosystem of hallmarks specific to PDAC promotes tumour survival, migration and continuously challenges clinical treatment strategies.^[8,9]

Tumour hypoxia has challenged radiotherapy for over 50 years, PDAC has one of the lowest recorded hypoxia readings as compared to many cancers.^[6] Radiotherapy for PDAC is traditionally controversial with contradictory clinical trial outcomes, such as the LAP02 2016 trail^[10] and the ESPACI study^[11] in which no improved overall survival and damage to organs at risk were observed, highlighting a need for further understanding and optimisation of this treatment for a disease with such dismal survival statistics.

Traditional pre-clinical models for studying PDAC treatments in vitro are often labelled misrepresentative with the failure of treatment advances for PDAC when reaching the clinical level. 2D cell culture is a fast and low cost approach to screen treatments, yet it fails to represent microarchitectures, environmental gradients and cell-cell/cell-matrix interactions present in vivo that are responsible for treatment resistance. Moreover, the traditional use of xenografts allows realistic in vivo architecture but fails to represent realistic physiology, heterogeneity and mutation rates along with challenges of variability and high cost. ^[7-9]Thus there is a need for a reliable, low cost and representative system to test delivery strategies. The interdisciplinary field of tissue engineering is advancing to bridge the gap between simplistic 2D cell culture models and complex xenografts, more readily emulating tumour architecture, porosity, cell-cell and cell-matrix interactions, as well as hypoxic regions.^[1] More specifically, tissue engineering allows mimicry of bio-physical-chemical and mechanical properties of the tumour microenvironments in a finely tuned in vitro setting, facilitating an animal free and cost effective platform as a representative tool for treatment screening and resistance profiling for cancer research ^[2]. Furthermore, 3D models are emerging as useful tools for radiation screening for PDAC ^[12] and are required for advancing radiation testing ^[13].

The BioProChem Group at the University of Surrey have previously fabricated a 3D porous polymeric scaffolding system to support long term PDAC cell growth and in vivo properties such as dense cellular masses, initiated collagen-1 growth and the development of hypoxic regions ^[1-2] for the application chemo-radiotherapy screening ^[3]. In addition, the research group produced the first scaffold assisted multicellular model for PDAC ^[14]. Utilising the 3D porous polymeric scaffolding system, here we aim to replicate the PDAC TME to investigate hypoxia-induced radio-resistance.

Materials and Methods:

Fabrication of polymeric 3D scaffolds employed the Thermally Induced Phase Separation method (TIPS) ^[1-3]. PANC-1 cells were seeded at 0.5x10⁶ and cultured for 4 weeks before being in a hypoxic chamber (5% oxygen). Radiation exposures were performed using an orthovoltage X-ray unit at the Royal Surrey County Hospital. Samples were characterised at 24 hour, 3 days and 7 days post radiation. Confocal laser scanning microscopy (CLSM) of multiple scaffold sections, enabled scaffold characterization, allowing analysis of cellular organisation and viability and mapping of (radio-)resistance. More specifically, staining of live/dead, Caspase 3/7, HIF, actin, collagen and Dapi allowed characterisation of cellular and ECM response to hypoxia (5% O2) and radiation (6 Gy).

Results and Discussion:

This research provides a platform for radiation responses in a 3D hypoxic PDAC system. These data represent changes in live/dead and apoptotic cell profiles with 0 Gy and 6 Gy radiation treatment at 21% O₂ and 5% O₂ at various time points (24hr, day 3 and Day 7 post treatment). Generally, low oxygen levels lead to high treatment resistance. Moreover, changes in hypoxic biomarkers and ECM proteins were identified. These data show for the first time (i) a 3D polymeric scaffold supporting long-term hypoxic PDAC cell culture (ii) a long-term post-treatment in situ cell characterisation for radiotherapy treatment patterns. Overall, this system provides a versatile platform to study hypoxia-associated radio-resistance profiling of PDAC.

Significance and Impact:

PDAC is a cancer of unmet clinical needs. Traditional preclinical research techniques often fail to emulate treatment responses within the clinic, resulting in the failure of advancing treatments. The treatment resistant nature of PDAC requires the development of more effective treatments. This calls for a realistic replica of the unique and extremely hypoxic TME for treatment screening and resistance profiling. This novel hypoxic 3D PU scaffold acts as a platform for biomimicry of the complex and hypoxic PDAC TME. This is the first 3D PU hypoxic scaffold system for PDAC radiation treatment studies.

Acknowledgements:

This research was supported by the Department of Chemical and Process Engineering of the University of Surrey as well as the National Physical Laboratory, the EPSRC and the Royal Society. E.V. is grateful to the Royal Academy of Engineering for an Industrial Fellowship.

References:

[1] Totti, S., et al. (2018). RSC Advances, 8(37).

[2] Totti, S., et al. (2017). Drug Discovery Today, 22(4).

[3] Gupta, P., et al. (2019). RSC Adv., 9(71),

[4] American Cancer Society. (2019). Cancer Facts and Figures 2019.

[5] Cascinu, S. et al. (2010). Ann. Oncol, 21, 55–58.

[6] O’Reilly, D., et al. (2018). Pancreatology, 1–9.

[7] Rahib, L., et al. (2014). Cancer Research, 74(11), 2913–2921.

[8] Xie, D., & Xie, K. (2015). Genes & Diseases, 2(2), 133–3.

[9] Pickup, M., et al. (2014). EMBO Reports, 15(12), 1243–1253.

[10] Hammel, P., et al. (2016). JAMA, 315(17), 1844.

[11] Neoptolemos, J. P., et al. (2004). New England Journal of Medicine, 350(12), 1200–1210.

[12] Wishart, G., et al (2021). British Journal of Radiology, 20201397.

[13] Mohajer, J. K., et al (2018). British Journal of Radiology, 20180484.

[14] Gupta, et al. Frontiers in Bioengineering & Biotechnology. 2020; 8 (290).